Ionotropic glutamate receptors (iGluRs) are membrane proteins which act as molecular pores and mediate signal transmission at the majority of excitatory synapses in the mammalian nervous system. The 7 gene families of ionotropic glutamate receptors (iGluRs) in humans encode 18 subunits which assemble to form 3 major functional families named after the ligands which were first used to identify iGluR subtypes in the late 1970s: AMPA, kainate and NMDA. Because of their essential role in normal brain function and development, and increasing evidence that dysfunction of iGluR activity mediates multiple neurological and psychiatric diseases, as well as damage during stroke, a substantial effort in the Laboratory of Cellular and Molecular Neurophysiology is directed towards analysis of GluR function at the molecular level. Atomic resolution structures solved by protein crystallization and X-ray diffraction provide a framework in which to design electrophysiological and biochemical experiments to define the allosteric mechanisms underlying ligand recognition and the gating of ion channel activity. This information will allow the development of subtype selective antagonists and allosteric modulators with novel therapeutic applications and reveal the inner workings of a complicated protein machine which plays a key role in brain function. The recent crystallization of the ligand binding cores of AMPA, kainate and NMDA receptor subunits, and a related bacterial receptor from the photosynthetic bacterium syncheocystis PCC 6803 which we named GluR0, has revealed for the first time the molecular mechanisms underlying the binding of agonists and antagonists as well as providing insight into the mechanisms of activation and desensitization. During the past year experimental efforts in structural biology have been directed towards studies on the ligand binding cores of members of the kainate receptor gene family, and on the cytoplasmic domains of NMDA receptors. Although a large number of glutamate receptor agonist and partial agonist structures has been solved, relatively few antagonist complexes have been crystallized. This is likely the result of the different conformations of the closed cleft agonist bound structure and the open cleft antagonist bound structure and differences in mobility of the ligand protein complex. Agonists bound complexes are like rocks, crystallize easily, and have similar temperature factors in domain 1 and 2. Antagonist structures are hard to crystallize. High resolution structures of two novel GluR5 selective antagonists, UBP302 and UBP310, reveal a hyper extended conformation in which the ligand forces the domains to separate to a greater extent than observed in the GluR2 apo structure. Strikingly, electron density was excellent for domain 1, but less well defined for domain 2. Refinement of the final structure required TLS analysis and reveled both domain breathing motions and a higher overall mobility of domain 2. The structure of the UBP302 and 310 complexes is strikingly different from those previously solved for the GluR2 DNQX and NR1 5,7DCKA complexes and revealed a mode of ligand binding that could not be modeled from prior structural information. It is anticipated that this new information will facilitate the design of new subtype kainate receptor antagonists. Despite overall high amino acid sequence homology and similar gating kinetics, the exchange of single amino acids which differ between the AMPA and kainate receptor subtypes of iGluRs lead to identification of a non desensitizing AMPA receptor L483Y construct that during the past 5 years has been widely used as a tool to investigate numerous aspects of iGluR biology. The availability of similar constructs for kainate receptors would be of great interest, yet surprisingly paradoxical results have been obtained when mutations were introduced into the ligand binding core dimer interface of kainate receptors. To address this the GluR5 ligand binding core was crystallized in a dimeric assembly similar to that observed for AMPA receptors, and its structure determined at 2.1 ? resolution. Using this structure as a template a series of mutants was designed in the dimer interface with the goal of stabilizing the active conformation. These were also crystallized and their structures solved, and the sedimentation behavior of the purified ligand binding cores examined by analytical ultracentrifugation. We predicted that those mutations which stabilized kainate receptor dimer formation would attenuate desensitization. This was tested by rapid perfusion experiments using HEK293 cell patches which revealed complete block of GluR6 desensitization for constructs which stabilize the dimer assembly. Of interest for another construct which was designed to reproduce the local dimer structure of the GluR2 L483Y mutant, there was no block of desensitization or change in sedimentation behavior, even though when crystallized the resulting structure closely resembled the L483Y parent. Such results illustrate the importance of using a wide range of biophysical approaches to characterize receptor function, since although crystal structures capture in exquisite detail the molecular details of protein assembly, they cannot at present be used to reliably estimate the energetics of assembly of macromolecular complexes.